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Regular Article |
a Department of Biology, University of Milano, 20133 Milano, Italy
b Department of Biotechnology and Biosciences, University of Milano-Bicocca, 20126 Milano, Italy
ABSTRACT
The MSJ-1 gene encodes a murine DnaJ homologue that is expressed specifically in adult testis. DnaJ proteins act as cochaperones of Hsp70 proteins in promoting diverse cellular functions. In this study we used recombinant MSJ-1 proteins to produce MSJ-1 antiserum and to carry out in vitro binding assays. In a wide immunoscreening of mouse tissues, affinity-purified MSJ-1 antibodies recognize a unique protein of 30 kDa in male germ cells only. MSJ-1 is able to interact with the testis-specific Hsp70-2 protein and can be coimmunoprecipitated with Hsp70-2 from spermatogenic cells; binding of these two chaperones is consistent with the presence of a third component, which is so far unknown. MSJ-1 is weakly detected in early round spermatids, and its protein content increases in cytodifferentiating spermatids where it colocalizes with the developing acrosome and their postnuclear region. Hsp70-2, which is known to be highly expressed in meiotic cells, shows a subcellular localization in late differentiating spermatids that overlaps that of MSJ-1. MSJ-1 is also maintained in testicular and epididymal spermatozoa, where it sharply demarcates into two distinct cell areas; the outer surface of the acrosomal vesicle, and the centrosomal area. On the whole, our findings are consistent with a role for MSJ-1 in acrosome formation and centrosome adjustment during spermatid development, whereas its presence in mature spermatozoa suggests a special function during fertilization, shortly afterward, or both.
fertilization, spermatid, spermatogenesis, stress
INTRODUCTION
An essential cellular machinery is that constituted by a group of specialized proteins, known as molecular chaperones, that promote the folding and assembly of nascent proteins, the transport of proteins into cell organelles, and the assembly and disassembly of mature protein complexes which are involved in cell signaling or other regulatory processes that require subtle protein rearrangements or conformational changes. The most abundant family of molecular chaperones consists of the highly conserved 70-kDa heat shock proteins (the Hsp70s), which were originally identified by their induction under conditions of stress [1] and were also shown to be essential in numerous cellular processes under normal physiological conditions [2–4]. The apparent discrepancy between the broad Hsp70 substrate specificity on the one hand, and the high specialization of Hsp70 function on the other, has been explained by the discovery that Hsp70s work in concert with partner cochaperones, the DnaJ proteins [5, 6]. The DnaJ proteins are believed to customize Hsp70s, that function basically in binding unfolded polypeptides, for specific roles as that in protein traffic, gene regulation, uncoating of clathrin-coated vesicles, and so on [7]. Moreover, DnaJ proteins, as opposed to Hsp70s, constitute a heterogeneous group of multidomain proteins [8] defined by the highly conserved J domain, which has been found necessary for stimulation of interacting Hsp70 ATPase activity [9]. Through molecular cloning, we recently identified a new DnaJ family member [10], which we called MSJ-1, for first mouse spermatogenic cell DnaJ protein, on the basis of its pattern of expression, which is restricted to male germ cells only. More recently, it has also been termed HSJ-3 [8]. MSJ-1 possesses the fingerprinting J domain and the adjacent glycine/phenylalanine-rich sequence, but it lacks the cysteine-rich zinc finger domain and has no organelle-targeting sequence or posttranslational modification motif [10]. In CREMt-deficient mice [11] the expression of MSJ-1 was found to be strongly reduced; this information, together with in situ hybridization findings, suggested that MSJ-1 could be a haploid phase-specific gene product with a possible CRE element in its promoter [12].
Spermiogenesis is a highly specialized process of cell morphogenesis that results in extensive remodeling of haploid round spermatids in the generation of flagellate spermatozoa. This occurs by the activation of sets of haploid-specific genes, which leads to the appearance of novel proteins that are involved in the construction of peculiar structures and in cell remodeling [13]. By its features, the MSJ-1 gene product may have a role during spermiogenesis and represent the chaperone partner of a spermatogenic cell-specific HSP70, as Hsp70-2 [14, 15] or Hsc70t [16]. So, to better characterize MSJ-1, we produced recombinant proteins to obtain specific antibodies against MSJ-1 and performed in vitro and in vivo protein interaction assays. A 30-kDa protein was specifically immunorecognized in testis and was found able to interact with Hsp70-2. Immunocytochemistry revealed an intriguing localization of the protein, which during the morphogenetic process of spermiogenesis becomes confined to peculiar cellular districts, and is maintained in mouse epididymal spermatozoa.
MATERIALS AND METHODS
Production and Purification of Recombinant Proteins
An MSJ-1 cDNA fragment, encoding essentially the C-terminal portion of MSJ-1 (aa 145–242) [10], was cloned in frame with glutathione S-transferase (GST) using the pGEX-4T-1 vector (Amersham-Pharmacia, Little Chalfont, England). Full-length MSJ-1 cDNA, obtained in our laboratory, was also cloned in frame to GST using the pGEX-1 vector (Amersham-Pharmacia). Recombinant proteins, that is, the fusion protein GST/MSJ1(145–242) and the fusion protein GST/full-length MSJ1 (GST/MSJ1), were expressed in Escherichia coli DH5
and purified onto glutathione-sepharose beads (Amersham Pharmacia) following standard procedures. The purity of the GST fusion proteins was judged by SDS-PAGE and Coomassie blue-staining. The protein concentration was assessed by comparison with BSA standards using a Bio-Rad (Hercules, CA) DC protein assay.
MSJ-1 Antibodies
GST/MSJ1(145–242) protein was used to immunize New Zealand rabbits. To characterize the antiserum, a series of Western blot assays were carried out by testing in duplicate (immune serum/preimmune serum) blotted protein samples (fusion protein and its thrombin [Sigma, St. Louis, MO] digests plus mouse testis homogenate) with serial dilutions of the antiserum. Polyclonal antibodies were then affinity-purified using a GST-coupled Affigel-10 column (Bio-Rad) as described elsewhere [17].
Animals, Tissues, and Sperm Cell Collections
CD-1 mice were used. Tissue homogenates were obtained immediately after dissection as previously described [18]. For testis developmental studies, the gonads were collected from animals at Day 7, 16, 23, or 35 of neonatal life. Spermatogenic cell suspensions were obtained essentially according to the method of Bellvé [19] and postmeiotic cells were isolated as reported previously [20]. Epididymal spermatozoa were recovered from freshly excised epididymides as described elsewhere [18].
In Vitro Binding Studies
For GST pull-down assays, spermatogenic cell lysates were obtained by resuspending the pellets of mixed spermatogenic cell preparations (1 x 107 cells) in lysis buffer (10 mM Tris-HCl pH 7.5, 100 mM NaCl, 1 mM EGTA, 5 mM benzamidine, 2 mM phenylmethylsulfonyl fluoride, 200 µM Na3VO4, and 100 µg/ml leupeptin). After sonication (three 5-sec bursts; a Bransonic sonifier set at 50 W), Triton X-100 was added to a final concentration of 0.1% and the samples were incubated for 20 min at 4°C with gentle shaking. The insoluble material was pelleted by centrifugation (13 000 x g, 20 min, 4°C) and discarded; the supernatant was the cell lysate. To the cell lysate (70 µl) 30 µl glutathione-sepharose beads were added, incubated for 15 min with gentle shaking, and then removed by centrifugation. The clarified lysate, after addition of ATP to a final concentration of 2 mM, was incubated with 5 µg of purified GST/MSJ1 or GST alone for 2 h at room temperature or overnight at 4°C, followed by 1 h of incubation with glutathione-sepharose beads. The complexed beads were recovered by centrifugation, washed four times with 50 mM Tris-HCl pH 8.0, 0.1% Triton X-100, 1 mM EGTA, 5 mM benzamidine, 10 mM Na4P2O7, 400 mM Na3VO4, and 10 µg each of leupeptin and aprotinin per milliliter, and solubilized in SDS-PAGE sample buffer for resolution by subsequent SDS-PAGE and analysis by Western blotting.
For Far-Western experiments, spermatogenic cell lysates were fractionated by SDS-PAGE and transferred to a nitrocellulose membrane (Amersham-Pharmacia) using the blotting buffer without methanol. Membranes were denatured in 7 M guanidine-HCl dissolved in 50 mM Tris-HCl pH 8.2, 50 mM dithiothreitol, 2 mM EDTA, and 0.25% nonfat milk (NFM) for 1 h at room temperature; then renatured in 50 mM Tris-HCl pH 7.5, 100 mM NaCl, 2 mM dithiothreitol, 2 mM EDTA, 0.1% Nonidet P-40, and 0.25% NFM overnight at 4°C. After incubation in the blocking solution containing 5% NFM for 2 h at room temperature, membranes were incubated with GST/MSJ1 or GST alone (8 µg/ml) for a further 2 h. To assess direct interactions between MSJ1 and electrophoresed proteins, bound fusion proteins were detected by a rabbit anti-GST antibody (Santa Cruz Biotechnology, Santa Cruz, CA) followed by a horseradish peroxidase-conjugated anti-rabbit antibody (Santa Cruz Biotechnology) and a chemiluminescence system (Pierce, Rockford, IL).
Immunoprecipitation and Immunoblotting
Freshly prepared lysates from suspensions of mixed spermatogenic cells or isolated spermatids [20], first preadsorbed to protein A-sepharose beads (Amersham-Pharmacia) and then clarified by centrifugation, were processed for immunoprecipitation or coimmunoprecipitation assays. For immunoprecipitation, lysates were mixed with the appropriate antibody (anti-MSJ-1 or anti-Hsp70-2, a generous gift of M. Eddy, National Institutes of Health, Research Triangle Park, NC) for 2 h, followed by addition of protein A-sepharose beads for an additional hour at 4°C. Immunoprecipitates were washed twice with the ice-cold lysis buffer (20 mM Hepes-NaOH pH 7.2, 100 mM NaCl, 2 mM EDTA, 0.5% Nonidet P-40, 0.5% Triton X-100, 2 mM benzamidine, and 10 µg each of leupeptin and aprotinin per milliliter) and twice with the same buffer but without detergents. Then, the immunocomplexes were eluted by boiling for 5 min in 2x SDS sample buffer, resolved by SDS-PAGE, and transferred to nitrocellulose membranes to be immunoprobed with the immunoprecipitation antibody. For coimmunoprecipitation assays, slightly modified experimental conditions were used; lysates obtained in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Triton X-100, 1 mM EDTA, 2 mM PMSF, 2 mM benzamidine, and 10 µg each of leupeptin and aprotinin per milliliter were incubated first with ATP (2 mM final concentration) and then with Hsp70-2-antibody or preimmune serum, and incubated for 1 h. After a 30-min incubation in the presence of protein A-sepharose beads, immunoprecipitates were washed twice with the lysis buffer and once with the same buffer but without detergents, and then processed for SDS-PAGE and blot transfer. Membranes were then probed with MSJ-1-antibody.
Immunofluorescence Microscopy
Sexually mature testes were fixed at 4°C in buffered 4% paraformaldehyde and embedded in paraffin. Deparaffinized sections (7 µm) were processed for immunolabeling essentially as previously reported [18]. Spermatogenic cells and cauda epididymal spermatozoa, smeared on slides treated with 3-aminopropyltriethoxy-silane, were methanol-fixed and subjected to a double-fluorescent staining. First, the cells were immunostained with the indicated primary antibody (1:200 dilution in 3% BSA in a Tris-buffered saline), followed by 1:200 rhodamine-conjugated anti-rabbit immunoglobulin G (IgG; Sigma) or 1:400 Alexa 488-conjugated anti-rabbit IgG (Molecular Probes, Inc., Eugene, OR), as the secondary antibody. In control samples, primary antibody was omitted or replaced with preimmunization rabbit serum. Then, to localize nuclei, the DNA stain, 4,6-diamino-2-phenylindole (DAPI) was used (1 µg/ml). Cells were examined with an Olympus epifluorescence microscope equipped with standard filter sets for red (rhodamine), green (Alexa 488), and blue (DAPI) fluorescence, or with a confocal microscope equipped for red and green fluorescence (Leica TCS NT). Images from samples stained with rhodamine and DAPI were acquired with a scanner and elaborated with a personal computer.
RESULTS
Identification of MSJ-1 Protein by MSJ-1 Antiserum and Purified Antibodies
To obtain an antiserum specifically developed against MSJ-1, we produced a recombinant fusion protein, GST/MSJ1(145–242), which contains part of the middle portion and the carboxyl-terminal portion of MSJ-1, but not the J domain, which is believed to be highly conserved among all DnaJ proteins. Specificity of the antiserum was determined by immunoblot analysis using the recombinant protein (about 40 kDa) and its thrombin digests, which yield the GST moiety (28 kDa) and the MSJ(145–242) moiety (12 kDa). The results (Fig. 1B) showed that the antiserum reacted with the recombinant protein (lane d) and its thrombin digests (lanes b and c) plus the control GST protein (lane e), whereas the GST antibodies used in a parallel blot of controls (Fig. 1A) recognized the recombinant protein (lane d) and the GST moiety (lanes b, c, and e) only. Interestingly, a spermatogenic cell lysate yielded an immunoreactive signal when assayed with the antiserum (Fig. 1B, lane a), but not when probed with GST antibodies (Fig. 1A, lane a). This positive signal corresponds to a protein of about 30 kDa, a molecular weight that is in agreement with what we estimated for the predicted MSJ-1 protein [10]. Purified MSJ-1 antibodies were further prepared by depleting the antiserum of the anti-GST component. By comparing panels B and C in Figure 1, it is evident that the MSJ-1 antibodies we obtained were highly specific, because they recognized only the MSJ-1(145–242) moiety (Fig. 1C, lanes b and c) as well as the 30-kDa protein from the spermatogenic cell lysate (Fig. 1C, lane a). This last finding implies that MSJ-1 mRNA is effectively translated in mouse spermatogenic cells.
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MSJ-1 has been previously described as a novel testis-specific gene product because its transcript was found in testis only [10]. We screened a wide spectrum of mouse tissues through Western immunoblot analysis by using MSJ-1 antibodies. The results showed that a protein of 30 kDa was immunorecognized in testis only (Fig. 2A, lane b), whereas all other tissues, including ovary, were MSJ-1 immunonegative (Fig. 2A). So, the testis specificity of the MSJ-1 gene product is also confirmed at the protein level.
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Next, we carried out an immunoblot analysis with testis homogenates from mice at different postnatal ages to verify the appearance timing of MSJ-1 protein during spermatogenesis. MSJ-1 was detected only after round spermatids have already made their first appearance within the seminiferous epithelium (Fig. 2B, left panel, lanes c and d), which in the mouse, occurs at postpartum Day 20 [19]. Thus, MSJ-1 is haploid phase-specific. Moreover, when checked for the presence of MSJ-1, epididymal mouse spermatozoa were immunopositive, although to a lower extent compared with testicular germ cells (Fig. 2B, right panel, lanes a and b, respectively). This suggests that MSJ-1, which is maintained and not eliminated during spermiation, could have a role in sperm function.
MSJ-1/Hsp70-2 Protein Interaction
In mice, in addition to either the constitutive or stress-induced Hsp70 gene members, there are two testis-specific Hsp70 genes; the well-characterized Hsp70-2 [14, 15, 21], and the less-characterized Hsc70t [16, 22]. The Hsp70-2 gene is predominantly expressed in meiotic cells and the protein is abundant in unstressed spermatogenic cells from meiotic prophase until the end of spermatid development [14]. Whereas proteins that are able to associate with Hsp70-2 have already been identified in meiotic cells [15, 21], to our knowledge this has not yet been reported in postmeiotic cells. To examine whether MSJ-1 could interact with Hsp70-2, we performed a series of protein-protein interaction assays. Because the J domain of DnaJ proteins and, more specifically, the conserved sequence motif, HPD, which is also present in MSJ-1, are believed to be those implicated in the interaction with Hsp70 proteins [9, 23], we used the full-length MSJ-1 cDNA to construct a recombinant protein fused with GST (i.e., GST/MSJ1). The fusion protein was employed in GST pull-down assays in which spermatogenic cell protein lysates were mixed with GST/MSJ1 or control GST, and the bound proteins, after SDS-PAGE and blotting, were immunoprobed with the antiserum that was specifically developed against Hsp70-2 [14]. As shown in Figure 3A, spermatogenic Hsp70-2 was precipitated by GST/MSJ1 (lanes b and c), but not by the control GST protein (lane a); moreover, the addition of exogenous ATP in the pull-down assay resulted in a higher recovery of GST/MSJ1-precipitated Hsp70-2 (compare lane b with lane c). To determine whether the association between MSJ-1 and Hsp70-2 occurs directly, we performed Far-Western assays by probing electrophoresed spermatogenic cell proteins for their ability to bind GST/MSJ1. No interacting 70-kDa protein was immunodetected (data not shown). The Far-Western result suggests that the MSJ-1/Hsp70-2 interaction revealed by the GST pull-down assay is not direct, but rather is mediated by a third, so far unknown, component; a similar observation has already been reported for other DnaJ/Hsp70 protein complexes [24, 25].
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Next, we sought to ascertain whether the MSJ-1/Hsp70-2 association is physiological. Freshly prepared Hsp70-2 immunoprecipitates, obtained from either mixed spermatogenic cells or isolated spermatids, were assayed for the presence of coimmunoprecipitated MSJ-1. MSJ-1 was not detected in Hsp70-2 immunoprecipitates from cells lysed and washed in more stringent detergent conditions (see Materials and Methods section). In contrast, MSJ-1 was present in Hsp70-2 immunoprecipitates obtained under milder detergent (0.1% Triton X-100) conditions starting from both mixed spermatogenic cells (Fig. 3B, lane b) and isolated postmeiotic cells (Fig. 3B, lane c). As controls, immunoblotting with the homologous antibody confirmed the presence of Hsp70-2 (Fig. 3B, lane a), whereas MSJ-1 was not precipitated by preimmune serum (Fig. 3B, lane d). These findings suggest that the more stringent detergent conditions disrupted the Hsp70-2/MSJ-1 association; similar observations have already been reported for other protein-protein interactions [26].
Immunocytochemical Localization of MSJ-1 in Mouse Male Germ Cells
Testicular sections immunolabeled with MSJ-1 antibodies showed a positive reaction over the spermatid population of germ cells (Fig. 4A). Although immunostaining was present in round spermatids, it was most intense in late spermatids. To better visualize the cellular subtype or subtypes in which MSJ-1 is present and to determine its subcellular distribution, smears of spermatogenic cell suspensions were freshly prepared and processed for immunofluorescence microscopy. Samples were counterstained with the DNA intercalating dye DAPI, which by staining the cell nucleus, allows us to distinguish between round spermatids and cytoplasts. Figure 5 shows merged fluorescent images of spermatids at different stages of cytodifferentiation. Figure 5A is at low magnification and provides a general picture of MSJ-1 immunostaining; MSJ-1 is diffuse in the cytoplasm of round spermatids, with a preferential localization in the postnuclear region in early elongating spermatids, whereas in terminally differentiated spermatids (Fig. 5A, left bottom), MSJ-1 immunostains the acrosomal portion of the sperm head. The two spermatids in Figure 5B are at two distinct differentiation stages; MSJ-1 labels the postnuclear region of both spermatids, but in the riper one (top right), which has already acquired the characteristic nuclear shape of a rodent sperm cell, MSJ-1 also stains the developing acrosome. Figure 5C provides images of haploid male germ cells at further differentiation stages. In late elongating spermatids (left and right sides), MSJ-1 clearly localizes at the two opposite cellular poles; the falciform acrosomal region, which apically surrounds the hook-shaped nucleus, and the postnuclear region, most of which will give rise to the cytoplast, which is then excluded as a residual body from the fully formed testicular spermatozoon. In this regard, an MSJ-1 immunopositive/DAPI-negative detached residual body is visible in the upper center of the figure; this means that most protein has been lost in the last phases of spermiogenesis and explains the lower MSJ-1 content of mature spermatozoa revealed by immunoblot analysis. On the other hand, the round spermatid in Figure 5C (lower center), which is recognizable from the residual body by means of the double fluorescent staining, shows a very weak and patchy MSJ-1 immunolabeling; it is likely an early round spermatid (chromatin is not particularly condensed), the point at which MSJ-1 synthesis begins. This immunocytochemical analysis reveals that MSJ-1 localizes in sites that are important for both sperm morphogenesis and sperm function, so we further analyzed MSJ-1 at the confocal microscopy level. The laser scanning image acquired on a single focal plane set deep inside the acrosome of an elongating spermatid (Fig. 6, A and B) allows us to establish that MSJ-1 immunostaining is not acrosomal, but it defines the cytoplasmic outline of the acrosome on dorsal and ventral portions. Figure 6, C and D show MSJ-1 localization in epididymal spermatozoa. It can be seen that spermatozoa well preserve the acrosome-delimiting MSJ-1 immunolabeling and, in addition, exhibit an intense immunopositive spot localized to the neck region, which is consistent with the pericentriolar area, and which now, with the loss of the residual cytoplasm, is easily recognizable. Indeed, two orthogonally placed and MSJ-1-stained structures are clearly visible in the enlargement of Figure 6D.
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Spermatogenic cells were also Hsp70-2 immunostained. The protein is present in great amounts in spermatocytes and round spermatids [14] (and confirmed here). In late differentiating spermatids (Fig. 7) most Hsp70-2 accumulates in the cytoplasmic lobe that will give rise to the residual body, but a residual, fine immunolabeling is still detectable in the acrosomal portion of the head that delineates the dorsal and ventral outlines of the acrosomal surface (Fig. 7, A and C). This Hsp70-2 immunostaining overlaps that of MSJ-1 exhibited in spermatids at the same differentiation stage.
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DISCUSSION
MSJ-1, a novel member of the DnaJ family of proteins, was originally cloned from mouse spermatogenic cells [10]. DnaJ proteins play important regulatory roles as cochaperones, recruiting Hsp70 partners through their J domain; moreover, DnaJ proteins, by the way of their not-conserved protein region [9, 26, 27], can associate with cell-specific protein substrates giving rise to a ternary complex formed by Hsp70/DnaJ/protein substrate. When the MSJ-1 cDNA was identified [10], the predicted protein was not homologous to any other known DnaJ proteins, with the exception of HSJ-1, the human neuronal DnaJ protein [28], which shares a 48% amino acid sequence identity in a 175-amino acid overlap. More recently, two novel DnaJ proteins have been identified that share a greater than 50% amino acid sequence identity with MSJ-1; the murine Mrj [29] and the human HsJ-2 [30]. It is interesting that human HsJ-2 is expressed preferentially in the testis, where it undergoes a developmentally regulated expression and binds to the pituitary tumor-transforming gene protein (PTTGp), and gives rise to a protein complex that has been suggested to have a role in male germ cell differentiation.
In this study, we showed that MSJ-1 is expressed as a 30-kDa protein, a molecular weight that is in agreement with that calculated from its cDNA sequence. By developing an antiserum against a recombinant GST/MSJ protein, we have obtained affinity-purified MSJ-1 antibodies that are highly specific. When used in Western blotting for a wide immunoscreening of mouse tissues, these antibodies recognized a unique protein of 30 kDa in testis only and, more specifically, in spermatogenic cells isolated from sexually mature but not puberal testis, and in mature spermatozoa. These findings indicate that MSJ-1 mRNA is effectively translated in postmeiotic male germ cells, an observation that is not so obvious because evidence is accumulating that testis-specific transcripts show differential abilities for translation and some remain untranslated [31]. Moreover, these findings extend previous work [10, 12] demonstrating the presence of MSJ-1 in mature spermatozoa. Our immunofluorescence study is in agreement with the postmeiotic appearance of MSJ-1 revealed by the testis developmental immunoblot. Premeiotic and meiotic cells are MSJ-1 immunonegative, whereas round spermatids show a weak and patchy MSJ-1 distribution that early on becomes confined to the postnuclear area. In spermatids with condensing chromatin and developing acrosome, MSJ-1 is present in higher amounts and localizes in the acrosomal region, as well as in the postnuclear region. In terminally differentiated spermatids, MSJ-1 clearly marks the acrosome, and confocal laser microscopy has revealed that this localization is not intra-acrosomal, but it surrounds the acrosome. Epididymal spermatozoa are still MSJ-1 immunopositive, maintaining periacrosomal localization and exhibiting a centrosomal MSJ-1 localization that was previously masked by the strong MSJ-1 immunopositivity of the cytoplasmic lobe. So, during the differentiation of male germ cells, MSJ-1 is docked to two cell sites that are significant for both sperm cytomorphogenesis and sperm function.
A role of the DnaJ proteins is to customize Hsp70s for functions related to protein traffic and translocation of proteins across membranes [3, 7]. Here we show that MSJ-1 is able to interact with the male germ cell-specific Hsp70-2. This interaction is consistent with the presence of a third component because it was revealed by two assays that allow detection of both direct and indirect protein associations—an in vitro assay, the GST pull-down, and an in vivo assay, coimmunoprecipitation—but it was not revealed by the Far-Western analysis, which is specific for direct protein interactions. Moreover, a yeast two-hybrid test carried out with MSJ-1 cDNA as a bait to examine possible direct Hsp70-2/MSJ-1 interaction was negative (M. Eddy, personal communication). The third component of the protein complex could be a substrate protein that binds to MSJ-1 on its own and is targeted by MSJ-1 to Hsp70-2 in its ATP-bound state, in a manner that is reminiscent of what has already been reported for other mammalian DnaJ/Hsp70 complexes [26]. In mouse spermatocytes, Hsp70-2 immunostains synaptonemal complexes [15] and functions as a molecular chaperone for the assembly of an active CDC2/cyclin B1 complex [21]. To our knowledge there are no reports of a specific role for Hsp70-2 in mouse spermatids; however, Mori et al. [32] recently reported that the frequency of acrosome-containing cells in Hsp70-2 null mice is less than 0.01% of that in wild-type mice. Here we show that in differentiating spermatids the localization of Hsp70-2 overlaps that of MSJ-1. Indeed, a large part of Hsp70-2 is confined to the cytoplasmic lobe that will then be lost; but Hsp70-2 is also present, in low amounts, in the periacrosomal region. This suggests that during spermiogenesis Hsp70-2 and MSJ-1 could work in concert by promoting either the assembly of periacrosomal material, or the targeting of anchored proteins to the cytoplasmic surface of the acrosomal membrane, or by importing proteins into the acrosome. Either way, each of these processes is extremely important, considering the fundamental role of a functional acrosome.
Moreover, MSJ-1 also stains the centrosomal area. This is another important site of sperm morphogenesis and function. During spermiogenesis the pair of centrioles migrate and attach to the base of the nucleus where the distal centriole, which is associated with pericentriolar satellite appendages, functions as a basal body for the developing 9+2 axoneme of the sperm flagellum. The mechanisms of protein transport, incorporation, and turnover in flagellar axonemes are still unknown; cellular Hsp70s, however, have been localized to the centrosome [33] and molecular chaperones have been indicated to play important roles in axonemal protein dynamics [34]. Moreover, there is some disagreement in the literature about the fate of the centrosomal material at the end of spermiogenesis in rodents [35, 36]; this is an important question to be solved in view of the debate on maternal or paternal inheritance of the centrosome during fertilization [37]. Actually, we can affirm that MSJ-1 maintains its periacrosomal and centrosomal localization in mature mouse spermatozoa. So, as our study suggests, MSJ-1 could interact with Hsp70-2 during spermiogenesis in promoting cytomorphogenic events; in spermatozoa this unique DnaJ protein may have other roles, such as involvement during fertilization by interacting with other Hsp70 proteins present in sperm cells [22] to promote the assembly of a protein signaling complex. In this regard, the function of DnaJ proteins in the maturation and activation of steroid hormone receptors has now been well established [6, 38–40] and, in addition, the stimulatory actions of progesterone on mammalian spermatozoa have been known for many years [41–43]. Work on examining the possible function of MSJ-1 during mouse fertilization and tracking its fate in the fertilized egg is in progress in our laboratory.
ACKNOWLEDGMENTS
We thank Dr. L. Perego (Milan, Italy) for constructing the recombinant protein used to produce the antiserum, Drs. B. Borgonovo and U. Fascio (Milan, Italy) for their technical assistance with confocal microscopy, and Dr. M. Eddy (National Institutes of Health, Research Triangle Park, NC) for the generous gift of Hsp70-2 antiserum.
FOOTNOTES
First decision: 9 January 2001.
1 This work was supported by the Cofin98 grant from MURST, Rome, to G.B. 
2 Correspondence: Giovanna Berruti, Department of Biology, University of
Milan, Via Celoria 26, 20133 Milano, Italy. FAX: 39 02 26604361; giovanna.berruti{at}unimi.it
Accepted: March 22, 2001.
Received: December 8, 2000.
REFERENCES
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